The present invention relates to materials and methods for the controlled expression of polynucleotides in plant cells.
Biological methods for the production of economically valuable compositions of matter in the form of polypeptides have shown promise as alternatives to traditional chemical syntheses. Although several biological systems have been successfully explored as potential sources of polypeptides, including proteins, each system has been found to have its limitations.
The simplest and most thoroughly investigated biological methods for chemical production are microbiological systems. Primitive prokaryotic cells have been amenable to investigation and have been found to produce a variety of small organic and inorganic compounds, as well as a variety of complex biomolecules, such as homologous and heterologous polypeptides and proteins. The most extensively characterized prokaryote, Escherichia coli, synthesizes complex biomolecules using a relatively straightforward process of gene expression requiring minimal expression control elements and an uninterrupted coding region. Further, genetic elements encoding heterologous polypeptides can be introduced and expressed in E. coli without much difficulty. With these advantages, a wide variety of polypeptides have been expressed in a controlled manner in this organism. However, E. coli cultures do require the costly inputs of energy and nutrients. The organism also does not readily secrete the produced polypeptides, adding to the time and expense required to isolate the desired compound. Although other microbes, e.g., species of Bacillus, do secrete polypeptides into growth media, cultures of these organisms also require costly inputs of energy and nutrients. Moreover, all of these primitive prokaryotic systems exhibit additional shortcomings such as the expensive effort to avoid culture contamination and the inability of the microbes to properly process or derivatize many expressed polypeptides to fully biologically active forms.
Yeast and fungi are fairly primitive eukaryotic cells that have also been used to produce polypeptides, including heterologous polypeptides. Although these cells may do a better job of reproducing the natural derivatization of most commercially desirable (i.e., eukaryotic) polypeptides, the reproduction is imperfect. Additionally, cultures of yeast or fungal cells are susceptible to contamination and the cells themselves require valuable resources in the forms of energy and nutrients, Efforts to obtain the desired chemicals, such as heterologous polypeptides, are also burdened by the frequent need to extract the chemical from the cell and purify that compound from the chemically complex contents of the yeast cell released during extraction.
Animal cells, e.g. mammalian cells, although expected to closely approximate the native derivation of many important polypeptides (e.g., human polypeptides), are very costly to culture, due to their sensitivity to contaminants, their requirements for energy, gases, and nutrients, and their limited lifespans. Isolation of the produced chemicals also would be it relatively expensive in view of the typical inability of mammalian cells to secrete products and the relative chemical complexity of the intracellular environment of these cells.
Plants, as photoautotrophic organisms, provide an alternative to heterotrophic animals as life forms for the production of chemicals. Transgenic plants have been generated, albeit typically to improve the characteristics of the plants themselves (e.g., to confer resistance to disease, to improve the yield of edible foodstuffs). Nevertheless, some transgenic plants have been used to produce chemicals such as heterologous polypeptides. Some expression control sequences (e.g., regulatory elements, signal peptide sequences) have been found to function in plant cells, or to preferentially function in the cells of particular plant tissues and organs. For example, Sijmons et al. (U.S. Pat. No. 5,650,307) expressed Human Serum Albumin (HSA) by fusing the HSA coding region to the leader sequence from Alfalfa Mosaic Virus. This fused coding region was placed under the control of the Cauliflower Mosaic Virus 35S promoter and the Nopaline Synthase terminator. The HSA was expressed in transgenic potato plants and transgenic tobacco cells. Sijmons et al. further disclosed the secretion of HSA by potato plant cells and recovery of the heterologous HSA from the intercellular space of those plants. Of course, this recovery method involved the destruction of the potato plants.
U.S. Pat. No. 5,580,768 also discloses the production and secretion of heterologous protein by a plant. In particular, the ""768 Patent discloses a transgenic rubber tree, with the expressed transgene protein being collected from wounds as a part of the latex. This system is highly specialized for use with Hevaea species (perennial tree species with slow growth), and the tree must be damaged by wounding to recover the heterologous polypeptide in the form of a latex mixture.
Transformed plant material has also been used to express heterologous polypeptides. Wongsamuth et al., Biotech. and Bioengineer. 54:401-415 (1997), report the use of hairy root cultures to express murine IgG1 monoclonal antibody. Further, some antibody activity was found in the medium of the hairy root cultures maintained under axenic conditions as heterotrophic biomasses requiring costly energy and nutrient inputs.
In plant expression systems, as in other biological expression systems, maximal utility is realized by an expression system that is controllable. Control of the timing and extent of polypeptide expression reduces the costs involved in maintaining the typically transformed host cells because recovery can be initiated at times that are suitable for the polypeptide being expressed. For example, recovery can be coincident with the period during which expression is elevated when attempting to produce and purify a labile polypeptide. To maximize the yield of stable polypeptides required in quantity, the recovery period may lag the expression period. In still other cases, the production of toxic polypeptides is delayed until optimal numbers of producing cells are present, with little, if any, lag in the recovery period.
A variety of controllable expression systems have been identified in animal, bacterial, yeast, or fungal cells, and some of these systems are also found in plant cells. However, the majority of these systems suffer from disadvantages in terms of the simple, versatile and economic production and recovery of polypeptides from plant cells. Frequently, the small molecule effector responsible for controlling expression is difficult to make or costly to obtain and, for those effectors that are available, problems associated with toxicity are frequently encountered. These toxicity considerations include the toxic potential of the effector on the host cell, as well as the deleterious presence of the effector in the isolated polypeptide preparation.
Phosphorus is a nutrient that plays a central role in energy metabolism and, in the form of phosphate, is found in the nucleic acids of all living organisms. Much of the phosphorus available in the environment is not in a bioavailable form such as orthophosphate, however. Consequently, diverse organisms have developed capacities for transforming environmental phosphorus into bioavailable forms that are assimilated. These capacities are evident in the number and diversity of genes that respond to phosphorus levels. One class of genes encodes phosphatase enzymes, which can generally be divided into alkaline and acid phosphatases. Within a given organism, there is variation in the number and characteristics of acid phosphatases that are expressed.
Acid phosphatase expression has been studied most extensively in lower organisms such as yeast, fungi and bacteria. Phongdara et al., Appl. Microbiol. Biotechnol. 50:77-84 (1998), characterized a yeast acid phosphatase sequence and reported that expression of the gene could be repressed by phosphorus. However, the host cell was H. polymorpha, a methylotrophic yeast subject to the limitations of culturing identified above. Similarly, Ferminan et al., Microbiol. 143:2615-2625 (1997), characterized an acid phosphatase gene in another yeast species. The use of an acid phosphatase 5xe2x80x2 control region to drive the expression of a heterologous gene has been reported for yeast (Kai et al., Seibutsu-Kogaku Kaishi 71:317-323 (1993), Ferminan et al., Appl. Environ. Microbiol. 64:2403-2408 (1998), Braspenning et al., BBRC 245:166-171 (1998), Shigematsu et al., J. Biol. Chem. 267:21329-21337 (1992)) and fungi (Macrae et al., Gene 132:193-198 (1993). The recombinant expression of bovine opsin, using an acid phosphatase signal sequence, has also been reported in yeast. Abdulaev et al., Protein Exp. Purif. 10:61-69 (1997). The expression of these acid phosphatase genes, and the use of acid phosphatase 5xe2x80x2 control regions to express heterologous genes, has all been done in yeast and fungi, however. Thus, a need remains for a controllable expression system freed of the culture requirements and other problems attending use of these lower eukaryotic cells.
In vascular plants (i.e., multicellular plants including gymnosperms, angiosperms, ferns and liverworts, among others), acid phosphatases have received less attention. A review by Duff et al., Physiol. Plantarum 90:791-800 (1994), stated that some Brassica nigra acid phosphatases were induced by phosphate starvation in the context of noting that a wide variety of environmental and developmental factors (e.g., plant hormones, flowering, and senescence, among others) influenced APase activity, emphasized the heterogeneity of plant APases in terms of subunit structure, kinetic properties, and localization, and concluded that the available data demonstrated a lack of understanding of the molecular events underlying APase induction. Working with Arabidopsis thaliana, Patel et al., Plant Physiol. 111 (2 Supp.):81 (1996), reported the sequence of an acid phosphatase gene (Genbank Acc. No. U48448), while Williamson et al., Plant Physiol. 97: 139-146 (1991) disclosed a sequence of an APase gene from tomato. In addition to reporting an A. thaliana APase gene sequence, Patel et al. noted that steady-state levels of transcripts rose during phosphate starvation. Consistent with that observation, Trull et al. reported that acid phosphatase expression in A. thaliana could be de-repressed by lowering phosphorus levels. Trull et al., Plant Physiol. 105 (1 Supp.): 112 (1994); Trull et al., Plant, Cell and Environ. 20:85-92 (1997). This de-repression could be mitigated in the oilseed rape through the use of the fungicide phosphonate. Carswell et al., Planta 203:67-74 (1997). However, none of these reports disclose or suggest the use of the 5xe2x80x2 control region of a vascular plant acid phosphatase to drive the expression of a heterologous gene in plant cells. Moreover, the A. thaliana acid phosphatase sequence (Genbank Acc. No. U48448) does not exhibit significant similarity to any lower eukaryotic acid phosphatase, confirming both the diversity of genes involved in phosphorus metabolism and the variation seen in the characteristics of acid phosphatase gene expression.
Thus, a need continues to exist in the art for a plant promoter capable of driving the expression of non-native coding regions in vascular plant cells, with that promoter being regulated by relatively simple, cost-effective techniques, such as controlling the bioavailability of phosphorus.
The present invention satisfies the aforementioned need in the art by providing a phosphorus-controllable plant promoter driving the expression of a non-native coding region (e.g., a transgene) in vascular plant cells. In the methods according to the invention, the non-native coding region is expressed in a plant cell, an intact plant portion, or a whole plant. Regulation of expression of the non-native coding region is achieved by controlling the level of at least one phosphorus compound delivered to a plant cell or by delivering a regulatory compound that affects a cell""s level of bioavailable phosphorus. Suitable regulatory compounds include a phosphorus-depriving agent (i.e., an agent that deprives a plant cell of a physiologically significant amount of phosphorus), such as a phosphorus-sequestering agent, as well as a phosphorus-releasing agent, an agent that influences phosphorus transport into or out of a plant cell or an organelle thereof, or an agent that alters the distribution of phosphorus in bioavailable and non-bioavailable forms. Any type of phosphorus-depriving agent known in the art may be used in methods according to the invention. The simple and inexpensive expression methods of the invention may be used with any of a wide variety of vascular plant cells.
One aspect of the invention is directed to a method for expressing a non-native coding region in a vascular plant cell comprising the following steps: (a) transforming a vascular plant cell with a polynucleotide comprising a plant promoter controllable by a phosphorus compound operably linked to a non-native coding region, and (b) expressing the coding region. A coding region encodes an RNA or a polypeptide. A related aspect of the invention further comprises the step of controlling the level of the phosphorus compound by contacting the vascular plant cell with a regulatory compound. Thus, the methods extend to the use of all combinations of plant promoters controllable by phosphorus compounds and coding regions other than the operable linkage of a promoter controllable by a phosphorus compound to its natural or native coding region.
Another aspect of the invention is an isolated polynucleotide comprising a plant promoter controllable by a phosphorus compound operatively linked to a non-native coding region. A preferred plant promoter for use in the method is an acid phosphatase promoter. Also preferred is a plant promoter derived from a vascular plant source (e.g., members of the Brassicaceae family of plants, such as Arabidopsis thaliana and Brassica juncea). For example, a preferred plant promoter is selected from the group consisting of polynucleotides having sequences set forth in SEQ ID NO:1 (A. thaliana acid phosphatase promoter region; Genbank Acc. No. U48448), or fragments thereof having a minimum of 40 nucleotides, a recognizable xe2x88x9210 region having at least 90% similarity to the xe2x88x9210 consensus sequence(s) of eukaryotic promoters (e.g., TATRATG, where R is either A or G), and an element involved in expression control that is affected by phosphorus levels. In addition, the invention includes polynucleotides that hybridize to such polynucleotides under hybridization conditions of 3xc3x97SSC, 20 mM NaPO4 (pH 6.8) at 65xc2x0 C., and washing conditions of 0.2xc3x97SSC at 65xc2x0 C.
Coding regions useful in the methods of the invention include polynucleotides from any source, natural or synthetic. The invention is not limited by the coding regions that may be operatively linked to promoters controllable by a phosphorus compound. Suitable coding regions encode animal RNAs or polypeptides, as well as variants, fragments and derivatives thereof The encoded products may be recovered for use outside the host plant cell (e.g., therapeutically active products) or they may alter the phenotype of the host plant cell (e.g., conferring disease resistance, the ability to survive or grow in the presence of particular substrates). Examples of such coding regions include polynucleotides derived from vertebrates, such as mammalian coding regions for RNAs (e.g., anti-sense RNAs, ribozymes, and chimeric RNAs having ribozyme structure and activity) or polypeptides (e.g., human polypeptide coding regions). Other coding regions useful in the inventive methods are derived from invertebrates (e.g., insects), plants (e.g., crop plants), and other life forms such as yeast, fungi and bacteria. The invention further contemplates any vector known in the art comprising a polynucleotide according to the invention.
Another aspect of the invention is a vascular plant host cell transformed with a polynucleotide according to the invention, regardless of whether such host cell is isolated or found in a plant material selected from the group consisting of an intact plant portion (e.g., a root, a shoot or a leaf) and a whole plant. Host cells according to the invention include the vascular plant host cells used in methods for producing coding region products according to the invention, as described below. A wide variety of floating, submerged, and soil-based plants are useful in the inventive methods, including monocots such as ryegrass, alfalfa, turfgrass, eelgrass, duckweed, and wilgeon grass as well as dicots such as tobacco, tomato, rapeseed, Azolla, floating rice, water hyacinth and any of the flowering plants. Additional plants capable of recombinant polypeptide expression also may be used in the methods of the invention. Presently preferred plant cells are derived from the Brassicaceae family of plants, including Brassica species, A. thaliana, and Nicotiana tabacum (i.e., tobacco).
As mentioned above, another aspect of the invention is a method for expressing a coding region product comprising the following steps: (a) transforming a vascular plant host cell with a polynucleotide selected from the group consisting of a plant promoter controllable by a phosphorus compound and a non-native coding region, and (b) maintaining the vascular plant cell under conditions that permit expression of a coding region operably linked to the promoter. Any of the sets of conditions known in the art, some of which are disclosed herein, is used. In one embodiment, a phosphorus-controllable plant promoter is transformed into a suitable plant host cell and the promoter becomes operably linked to a coding region in vivo. In another embodiment, a plant host cell is transformed with a non-native coding region that is operably linked in vivo to a promoter controllable by a phosphorus compound.
In some embodiments of the invention, recovery of the expressed product, such as an RNA or a polypeptide, is desired and is accomplished using any technique known in the art, including conventional invasive techniques that destroy plant tissue, such as vacuum infiltration and mechanical disruption. A preferred recovery technique avoids destruction of plant material by obtaining the expressed polypeptide from a plant exudate, in the case of whole plants or intact portions thereof, or secretions, in the case of isolated plant cells. In recovering polypeptides from plant exudates, the exudate is externally contacted with an aqueous medium to effect an admixture of the medium and the exudate in the process of recovery. The contacting step of the inventive processes may involve a culture system relying on soil-based cultivation or water-based cultivation such as hydroponics or aeroponics. The polypeptide is recovered from exudate, which may be root exudate, guttation fluid oozing from the plant as an exudate via leaf hydathodes, or other sources of exudate, regardless of xylem pressure. The contacting and recovering steps of the processes may be performed continuously or in a batch mode. In one embodiment, the polypeptide is expressed in, and recovered from, a plant portion. The plant portions for use in the processes of the invention are intact and living plant structures. These plant portions may be distinct plant structures, e.g., shoots, leaves, and roots. Alternatively, plant portions may be part or all of a plant organ or tissue, provided the material is intact and alive.